Tuberculosis (TB) is an ongoing global health problem, causing an estimated 1.5 million deaths in 2014. In spite of growing drug resistance, few new drugs are in the development pipeline. Functions required by pathogen and host for disease to be established and progress would offer a broad range of novel targets. However, our understanding of the relevant functions remains limited, necessarily limiting our ability to target them with new therapies. Upon infection with the bacterium that causes TB, Mycobacterium tuberculosis (Mtb), macrophage recognition of bacterial products triggers innate immune programs. One response induced following Mtb-mediated permeabilization of the phagosomal membrane is the type I interferon (IFN) response. ESX-1-mediated protein secretion, required for Mtb growth in macrophages and mice, was previously known to be required for phagosomal permeabilization. In recent work, we have determined that additional Mtb factors required for growth in the host are required to induce the type I IFN response, suggesting that loss of type I IFNs can serve as a surrogate marker for loss of critical bacterial functions. Whether type I IFNs play a causative role in TB pathogenesis or are simply a marker of phagosomal permeabilization is an area of active scientific debate, and small molecules that block this response would be useful tools for definitively answering this question. In the proposed work, we will (1) optimize a high-throughput assay to quantitate macrophage type I IFNs upon Mtb infection (2) apply that assay to a 50,000 compound library to identify small molecule inhibitors of the type I IFN response and (3) use secondary and tertiary assays to prioritize compounds for further study and development as potential anti-TB agents. Our screening assay will couple high-throughput ELISAs with imaging based quantitation of macrophage number to capture both type I IFN quantity and compound-related macrophage toxicity in the initial read-out. To prioritize compounds confirmed by retesting to be true hits, we will apply a series of secondary assays. We will first use in vitro growth assays to determine whether each compound has an effect on Mtb growing in culture alone. To determine which hits restrict Mtb intracellular growth, we will then use a high-content imaging assay to quantitate Mtb growth in compound- treated macrophages. We will then use a genetic tool to determine which hits impair Mtb-mediated phagosomal permeabilization. We will prioritize compounds for further study and development based on potency and toxicity, activity in secondary assays, predicted targets, and structural diversity. At the end of this work we anticipate having a series of small molecules that will serve both as tools to ask and answer critical questions about the role of the type I IFN response in TB infection and as starting points for the development of new anti-TB drugs targeting entirely novel bacterial and host functions.